Mass spectrometric methods and products

ABSTRACT

The invention involves assays, diagnostics, kits, and assay components for mass spectrometry and other methods to determine levels of glycated CD59 in subjects.

RELATED APPLICATIONS

The present application is a divisional of and claims priority under 35U.S.C. §120 to U.S. patent application, U.S. Ser. No. 12/917,271, filedNov. 1, 2010, which is a divisional of and claims priority under 35U.S.C. §120 to U.S. patent application, U.S. Ser. No. 11/794,635, filedSep. 11, 2009, now issued as U.S. Pat. No. 7,833,725, which is anational stage filing under U.S.C. §371 of international PCTapplication, PCT/US2006/000310, filed Jan. 6, 2006, which claimspriority under 35 U.S.C. §119(e) to U.S. provisional patent application,U.S. Ser. No. 60/641,762, filed Jan. 6, 2005; each of which isincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under grant numberCA087427 from the National Institutes Health (NIH). The Government hascertain rights in this invention.

FIELD OF THE INVENTION

The invention involves assays, diagnostics, kits, and assay componentsfor mass spectrometry and other methods to determine levels of glycatedCD59 in subjects.

BACKGROUND OF THE INVENTION

Diabetes Mellitus (diabetes) is a leading cause of morbidity andmortality in the adult population. This is primarily because diabeticpatients tend to develop vascular complications that involve the kidneys(diabetic nephropathy), the retina (diabetic retinopathy), as well aslarge and small blood vessels in other organs (macro- and micro-vasculardisease) including nerves (diabetic neuropathy). It is well establishedthat the vascular complications of diabetes are caused by elevated bloodglucose levels over long periods of time. Elevated blood glucose levelsaffect proteins by a process known as glycation. Different “glycated”proteins have been identified in diabetic subjects, including albumin,hemoglobin and others. Measurement of the extent of protein “glycation”of certain proteins is considered a valuable clinical tool to assesslong term glycemic control and thereby the efficacy of diabetestreatment.

Glycation, the non-enzymatic attachment of glucose to proteins, isconsidered a major pathophysiological mechanism causing tissue damage indiabetic subjects. Glycation involves the reaction of glucose and/orother reducing sugars with amino groups in proteins resulting in theformation of a Schiff base or aldimine. This labile adduct cantautomerize via the Amadori rearrangement to the more stable ketoamine.The function of the glycated protein may be impaired, depending on thelocation of the amino group(s) affected. For example, amino-terminalglycation of the β-chains of hemoglobin gives rise to the glycated tohemoglobins (HbA1) in which responsiveness to 2,3-diphosphoglycerate isdecreased and oxygen affinity increased. Glycation of the major thrombininhibitor of the coagulation system, antithrombin III, decreases itsaffinity for heparin, and has been postulated to contribute to thehypercoagulable state associated with diabetes.

Mass spectrometry has been used for the examination of protein glycationin diabetes research. Low- and high-resolution mass spectra, GC/MS,collisional activation spectroscopy, ESI, and MALDI/MS have been used inresearch settings for structural identification and quantitativeassessment of glycation end products and glycation of proteins.(Lapolla, A, et al., Mass Spectrometry Reviews, 2000, 19:279-304). Thesemethods can be time-intensive and the markers for assessing glycationlevels and related effects of increased glycation in diseases such asdiabetes are not optimal or applicable for rapid, reliable clinicalapplications.

In the clinical assessment of diabetes, protein glycation in diabeticsubjects is currently measured in blood by estimating the amount ofglycated hemoglobin (hemoglobin A1c) through a complicated clinical testthat requires extraction of a blood sample. Accordingly, there is a needfor a simplified, faster, and less invasive method for rapid monitoringof protein glycation levels.

SUMMARY OF THE INVENTION

The inventor has discovered highly accurate and reproducible methods forquantitating CD59 and glycated CD59 in biological samples. The methodsallow high-throughput, accurate analysis of samples, permitting a rapidand reliable measure for quantitating glycated protein in biologicalsamples. The inventor has also discovered novel products and kits. Theinvention relates, in part, to the determination of the relativeabundance of polypeptide products of the enzymatic digestion of glycatedCD59, non-glycated CD59, and glycated and non-glycated CD59 fragments.

The presence of soluble CD59 in bodily fluids, such as urine (˜5 μg/ml)and plasma (15-20 ng/ml), allow measurement of CD59 and glycated CD59 inthe fluids, and the invention permits analysis of large number ofsamples through high-throughput mode. It is known that trypsinhydrolyzes peptide bonds on the N-terminus of positively charged aminoacids such as K and R. Modification of amino acids, such as glycation ofthe amino acid side chain, can change the enzymatic digestion map ofproteins. We have determined that to glycated K41 on CD59 is no longerrecognized as nonglycated CD59 and have developed novel methods thatallow the determination of the level of glycated CD59 in biologicalsamples. The novel methods are based in part on the altered digestion ofglycated and non-glycated CD59. In one embodiment of the invention, themethod of determining the level of glycated CD59 in a sample is a massspectrometry method.

The glycation of the membrane protein known as CD59, a key regulator ofthe complement system, has been discovered to be involved in thepathogenesis of the vascular complications of diabetes. Glycated CD59has now been identified in human urine, blood, saliva, and otherbiological fluids (see U.S. Pat. No. 6,835,545). The amount of glycatedCD59 in human urine and other biological fluids correlates with glycemiccontrol and levels of glycated hemoglobin. Detection of glycated CD59 inthe urine and other biological fluids of diabetic patients can be usedto monitor glycemic control in diabetic patients, and to select subjectsfor therapy.

According to one aspect of the invention, methods of determining thelevel of glycated CD59 in a sample are provided. The methods includedigesting CD59 in a sample, identifying a peptide yielded by thedigestion of the CD59 in the sample, and quantitating the identifiedpeptide as a determination of the level of glycated CD59 in the sample.In some embodiments, the digestion is an enzymatic digestion. In someembodiments, the enzyme is trypsin. In some embodiments, the peptide isidentified using immunoassay, gel electrophoresis, NMR, Westernblotting, chromatography, or mass spectrometry. In some embodiments, thepeptide is identified using mass spectrometry. In some embodiments, thepeptide is quantitated by comparing the level of the peptide to thelevel of one or more additional peptides yielded by the digestion. Incertain embodiments, the peptide is quantitated by comparing the levelof the peptide to a control level. In some embodiments, the peptideidentified is a peptide with one or more labels. In certain embodiments,the one or more labels is selected from the group consisting of stableisotopes, fluorescent labels, radiolabels, enzyme labels, andluminescent labels. In some embodiments, the peptide identified is apeptide having an amino acid sequence set forth as AGLQVYNK (SEQ IDNO:8), CWK, FEHCNFNDVTTR (SEQ ID NO:9), or CWKFEHCNFNDVTTR (SEQ IDNO:10). In some embodiments, the invention also includes spiking thesample with an internal standard before digestion. In some embodiments,the internal standard has one or more labels. In some embodiments, theone or more labels is selected from the group consisting of stableisotopes, fluorescent labels, radiolabels, enzyme labels, andluminescent labels. In some embodiments, the internal standard is a CD59peptide. In some embodiments, the internal standard is a peptidecomprising the amino acid sequence set forth as AGLQVYNKCWKFEHCNFNDVTTR(SEQ ID NO: 15). In some embodiments, the one or more labels are at oneor more residues that correspond to A31 through K38 of mature CD59peptide. In some embodiments, the one or more labels is on an amino acidof the portion of the sequence set forth as SEQ ID NO:15 that isAGLQVYNK (SEQ ID NO:8). In some embodiments, the one or more labels onAGLQVYNK (SEQ ID NO:8) are at one or more residues that correspond toresidues A31, G32, L33, Q34, V35, Y36, N37, or K38 of mature CD59peptide. In some embodiments, the label is at the residue of the peptidethat corresponds to residue V35 of mature CD59 peptide. In someembodiments, the peptide has the amino acid sequence AGLQV_(d8)YNK (SEQID NO:6). In some embodiments, the one or more labels are at one or moreresidues that correspond to C39 through R53 of mature CD59 peptide. Incertain embodiments, the one or more labels is on an amino acid of theportion of the sequence set forth as SEQ ID NO:15 that isCWKFEHCNFNDVTTR (SEQ ID NO:10). In some embodiments, the one or morelabels are at one or more residues that correspond to residues C39, W40,K41, F42, E43, H44, C45, N46, F47, N48, D49, V50, T51, T52, or R53 ofmature CD59 peptide. In some embodiments, the label is at the residue ofthe peptide that corresponds to residue F47 of mature CD59 peptide. Insome embodiments, the peptide has the amino acid sequenceCWKFEHCNF_(d8)NDVTTR (SEQ ID NO:19). In some embodiments, the peptidehas the amino acid sequence CWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:20). Insome embodiments, the one or more labels is on an amino acid of theportion of the sequence set forth as SEQ ID NO:15 that is CWK. In someembodiments, the one or more labels is on an amino acid of the portionof the sequence set forth as SEQ ID NO:15 that is FEHCNFNDVTTR (SEQ IDNO:9). In some embodiments, the one or more labels are at one or moreresidues that correspond to F42 through R53 of mature CD59 peptide. Insome embodiments, the one or more labels on FEHCNFNDVTTR (SEQ ID NO:9)are at one or more residues that correspond to residues F42, E43, H44,C45, N46, F47, N48, D49, V50, T51, T52, or R53 of mature CD59 peptide.In certain embodiments, the label is at the residue of the peptide thatcorresponds to residue F47 of mature CD59 peptide. In some embodiments,the peptide has the amino acid sequence FEHCNF_(d8)NDVTTR (SEQ ID NO:7).In some embodiments, one label is on an amino acid of the portion of SEQID NO:15 that is AGLQVYNK (SEQ ID NO:8), and one label is on an aminoacid of the portion of SEQ ID NO:15 that is FEHCNFNDVTTR (SEQ ID NO:9).In certain embodiments, In some embodiments, the label on AGLQVYNK (SEQID NO:8) is at a residue that corresponds to residues A31, G32, L33,Q34, V35, Y36, N37, or K38 of mature CD59 peptide. In some embodiments,the label on FEHCNFNDVTTR (SEQ ID NO:9) is at a residue that correspondto residues F42, E43, H44, C45, N46, F47, N48, D49, V50, T51, T52, orR53 of mature CD59 peptide. In some embodiments, the labels are at theresidues of the peptide that correspond to residue V35 and F47 of matureCD59 peptide. In some embodiments, the labeled peptide isAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTR (SEQ ID NO:17). In some embodiments,the labeled peptide is TKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ IDNO:5). In some embodiments, the peptide is quantitated by comparing thelevel of the peptide determined in the spiked sample, the level of theinternal standard added to the sample, and the level of one or moreadditional peptides yielded by the digestion. In some embodiments, thepeptide is quantitated by comparing the level of the peptide to acontrol level. In some embodiments, the peptide quantitated is a peptidehaving the amino acid sequence set forth as AGLQVYNK (SEQ ID NO:8), CWK,FEHCNFNDVTTR (SEQ ID NO:9), or CWKFEHCNFNDVTTR (SEQ ID NO:10). In someembodiments, the peptide quantitated is labeled with one or more labels.In some embodiments, the one or more labels are selected from the groupconsisting of stable isotopes, fluorescent labels, radiolabels, enzymelabels, and luminescent labels. In some embodiments, the labeled peptideis AGLQVYNK (SEQ ID NO:8), or FEHCNFNDVTTR (SEQ ID NO:9). In someembodiments, the one or more labels are on one or more of the aminoacids of the sequence set forth as AGLQVYNK (SEQ ID NO:8). In certainembodiments, the label on AGLQVYNK (SEQ ID NO:8) is at a residue thatcorresponds to residues A31, G32, L33, Q34, V35, Y36, N37, or K38 ofmature CD59 peptide. In some embodiments, the labeled peptide isAGLQV_(d8)YNK (SEQ ID NO:6). In some embodiments, the one or more labelsare on one or more of the amino acids of the sequence set forth asFEHCNFNDVTTR (SEQ ID NO:9). In some embodiments, the label onFEHCNFNDVTTR (SEQ ID NO:9) is at a residue that correspond to residuesF42, E43, H44, C45, N46, F47, N48, D49, V50, T51, T52, or R53 of matureCD59 peptide. In some embodiments, the labeled peptide isFEHCNF_(d8)NDVTTR (SEQ ID NO:7). In some embodiments, the sample is afluid sample. In some embodiments, the fluid sample is blood, urine, orsaliva. In some embodiments, the sample is a tissue sample. In someembodiments, the sample is obtained from a subject. In some embodiments,the subject is diabetic. In some embodiments, the subject is atincreased risk of becoming diabetic. In certain embodiments, the subjecthas received treatment for regulating blood sugar levels. In someembodiments, the subject has not received treatment for regulating bloodsugar levels.

According to another aspect of the invention, methods of evaluating atreatment for regulating blood sugar levels are provided. The methodsinclude obtaining a first level of glycated CD59 from a first sampleobtained from a subject undergoing treatment for regulating blood sugarlevels, obtaining a second level of glycated CD59 from a second sampleobtained from the subject at least one day after obtaining the firstlevel, comparing the first level to the second level as an indication ofevaluation of the treatment, wherein the levels of glycated CD59 aredetermined by the methods set forth in any of the foregoing aspects andembodiments of the invention.

According to yet another aspect of the invention, methods of selecting atreatment for regulating blood sugar levels in a subject are provided.The methods include obtaining a level of glycated CD59 from a sampleobtained from the subject, and selecting the treatment for regulatingblood sugar levels in the subject based at least in part on the levelobtained, wherein the level of glycated CD59 is determined by themethods set forth in any of the foregoing aspects and embodiments of theinvention.

According to yet another aspect of the invention, methods for assessingonset, progression, or regression of a condition characterized byabnormal levels of glycated protein are provided. The methods include,obtaining a level of glycated CD59 from a sample obtained from asubject, and comparing the level to a control as an assessment of onset,progression, or regression of the condition, wherein the level ofglycated CD59 is determined by the method set forth in any of theforegoing aspects and embodiments, of the invention.

According to another aspect of the invention, compositions are provided.The compositions include an isolated peptide comprising an amino acidsequence set forth as AGLQVYNKCWKFEHCNFNDVTTR (SEQ ID NO:15), whereinthe sequence is labeled with one or more labels. In some embodiments,the isolated peptide has the amino acid sequence set forth asTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11). In some embodiments, thelabel is selected from the group consisting of stable isotopes,fluorescent labels, radiolabels, enzyme labels, and luminescent labels.In some embodiments, the one or more labels are at one or more peptidethat correspond to residues A31, G32, L33, Q34, V35, Y36, N37, K38, C39,W40, K41, F42, E43, H44, C45, N46, F47, N48, D49, V50, T51, T52, or R53of mature CD59 peptide. In some embodiments, the one or more labels ison one or more amino acids of the portion of the sequence set forth asSEQ ID NO:15 that is AGLQVYNK (SEQ ID NO:8). In some embodiments, theone or more labels is on one or more amino acids of the portion of thesequence set forth as SEQ ID NO:15 that is CWKFEHCNFNDVTTR (SEQ IDNO:10). In some embodiments, the one or more labels is on one or moreamino acids of the portion of the sequence set forth as SEQ ID NO:15that is CWK. In some embodiments, the one or more labels is on one ormore amino acids of the portion of the sequence set forth as SEQ IDNO:15 that is FEHCNFNDVTTR (SEQ ID NO:9). In some embodiments, the labelis at the residue that corresponds to residue V37 and/or F47 of matureCD59 peptide.

In some embodiments, the peptide has the amino acid sequenceAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTR (SEQ ID NO:17). In some embodiments,the peptide has the amino acid sequenceAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:16). In some embodiments,the labeled peptide is TKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ IDNO:5).

According to yet another aspect of the invention, compositions areprovided. The compositions include an isolated peptide having the aminoacid sequence set forth as AGLQVYNK (SEQ ID NO:8). In some embodiments,the peptide is labeled with one or more labels. In some embodiments, theone or more labels are selected from the group consisting of stableisotopes, fluorescent labels, radiolabels, enzyme labels, andluminescent labels. In some embodiments, the one or more labels are atone or more residues that correspond to A31 through K38 of mature CD59peptide. In some embodiments, the one or more labels on AGLQVYNK (SEQ IDNO:8) are at one or more residues that correspond to residues A31, G32,L33, Q34, V35, Y36, N37, or K38 of mature CD59 peptide. In someembodiments, the label is at the residue of the peptide that correspondsto residue V35 of mature CD59 peptide. In some embodiments, the peptidehas the amino acid sequence AGLQV_(d8)YNK (SEQ ID NO:6).

According to yet another aspect of the invention, compositions areprovided. The compositions include an isolated peptide having the aminoacid sequence set forth as FEHCNFNDVTTR (SEQ ID NO:9). In someembodiments, the peptide has one or more labels. In some embodiments,the one or more labels are selected from the group consisting of stableisotopes, fluorescent labels, radiolabels, enzyme labels, andluminescent labels. In some embodiments, the one or more labels are atone or more residues that correspond to F42 through R53 of mature CD59peptide. In some embodiments, the one or more labels on FEHCNFNDVTTR(SEQ ID NO:9) are at one or more residues that correspond to residuesF42, E43, H44, C45, N46, F47, N48, D49, V50, T51, T52, or R53 of matureCD59 peptide. In some embodiments, the label is at the residue of thepeptide that corresponds to residue F47 of mature CD59 peptide. In someembodiments, the peptide has the amino acid sequence FEHCNF_(d8)NDVTTR(SEQ ID NO:7).

According to another aspect of the invention, compositions are provided.The compositions include an isolated peptide comprising the amino acidsequence set forth as CWKFEHCNFNDVTTR (SEQ ID NO:10), wherein thepeptide has one or more labels. In some embodiments, the peptide has theamino acid sequence set forth as CWKFEHCNFNDVTTRL (SEQ ID NO:18). Insome embodiments, the one or more labels are selected from the groupconsisting of stable isotopes, fluorescent labels, radiolabels, enzymelabels, and luminescent labels. In some embodiments of any of theforegoing compositions, the one or more labels are at one or moreresidues that correspond to C39 through R53 of mature CD59 peptide. Insome embodiments of any of the foregoing compositions, the one or morelabels are at one or more residues that correspond to residues C39, W40,K41, F42, E43, H44, C45, N46, F47, N48, D49, V50, T51, T52, or R53 ofmature CD59 peptide. In some embodiments, the label is at the residue ofthe peptide that corresponds to residue F47 of mature CD59 peptide. Insome embodiments, the peptide has the amino acid sequenceCWKFEHCNF_(d8)NDVTTR (SEQ ID NO:19). In some embodiments, the peptidehas the amino acid sequence CWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:20).

According to yet another aspect of the invention, compositions areprovided. The compositions include an isolated peptide with the aminoacid sequence set forth as CWK. In some embodiments, the isolatedpeptide has one or more labels. In some embodiments, the one or morelabels are selected from the group consisting of stable isotopes,fluorescent labels, radiolabels, enzyme labels, and luminescent labels.In some embodiments, the one or more labels are at one or more residuesthat correspond to C39 through K41 of mature CD59 peptide. In someembodiments, the one or more labels are at one or more residues that tocorrespond to C39, W40, K41 of mature CD59 peptide.

According to yet another aspect of the invention, kits for determiningthe level of glycated CD59 in a sample are provided. The kits include apackage including a container containing an isolated peptide andinstructions for the enzyme digest of a sample spiked with the isolatedpeptide to determine the presence and/or level of glycated CD59 in thesample. In some embodiments, the enzyme digest is a trypsin digest. Insome embodiments, the isolated peptide is a CD59 peptide. In someembodiments, the CD59 peptide has one or more labels. In someembodiments, the one or more labels is selected from the groupconsisting of stable isotopes, fluorescent labels, radiolabels, enzymelabels, and luminescent labels. In some embodiments, the isolatedpeptide comprises the amino acid sequence set forth as CWKFEHCNFNDVTTR(SEQ ID NO:10) and has one or more labels. In some embodiments, the oneor more labels are at one or more residues of CWKFEHCNFNDVTTR (SEQ IDNO:10) that correspond to residues C39, W40, K41, F42, E43, H44, C45,N46, F47, N48, D49, V50, T51, T52, or R53 of mature CD59 peptide. Insome embodiments, the label is at the residue of CWKFEHCNFNDVTTR (SEQ IDNO:10) that corresponds to residue F47 of mature CD59 peptide. In someembodiments, the peptide has the amino acid sequenceCWKFEHCNF_(d8)NDVTTR (SEQ ID NO:19). In some embodiments, the peptidehas the amino acid sequence CWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:20). Insome embodiments, the isolated peptide has the amino acid sequence setforth as AGLQVYNKCWKFEHCNFNDVTTR (SEQ ID NO:15) and has one or morelabels. In some embodiments, the isolated peptide has the amino acidsequence set forth as TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11) and hasone or more labels. In some embodiments, the one or more labels are atone or more residues of that correspond to residues A31, G32, L33, Q34,V35, Y36, N37, K38, C39, W40, K41, F42, E43, H44, C45, N46, F47, N48,D49, V50, T51, T52, or R53 of mature CD59 peptide. In some embodiments,the label is at the residue that corresponds to residue V35 and/or F47of mature CD59 peptide. In some embodiments, the peptide has the aminoacid sequence AGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTR (SEQ ID NO:17). In someembodiments, the peptide has the amino acid sequenceAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:16). In some embodiments,the peptide has the amino acid sequenceTKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTR (SEQ ID NO:25). In some embodiments,the peptide has the amino acid sequenceTKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:5). In some embodiments,the label is at a residue of the peptide that corresponds to residueC39, W40, or K41 of mature CD59 peptide. In some embodiments, the kitalso includes a container containing trypsin. In some embodiments, thepeptide is lyophilized. In some embodiments, the peptide is packaged inan aqueous medium. In some embodiments, the kit also includes anantibody or antigen-binding fragment thereof that selectively binds aCD59 peptide.

According to yet another aspect of the invention, kits for determiningthe level of glycated CD59 in a sample are provided. The kits include apackage that includes a container containing trypsin and a containercontaining an isolated CD59 peptide, and a container containing anantibody or antigen-binding fragment thereof that specifically binds toa trypsin-digest fragment of the CD59 peptide, and instructions for useof the antibody or antigen-binding fragment thereof to determine thepresence and/or level of glycated CD59 in a sample. In some embodiments,the kit includes one or more CD59 peptides of any of the aforementionedembodiments or aspects of the invention.

These and other aspects of the invention will be described in greaterdetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a kit according to the invention.

FIG. 2 is a graph of the total ion current of the peptide.

FIG. 3 is a mass spectrometry profile showing the peptides of interest.(AGLQV is SEQ ID NO:12; QVYNK is SEQ ID NO:13; and FEHCNFNDVTTR is SEQID NO:9).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 Amino acid sequence of full-length CD59 prior to removal ofthe signal peptide.SEQ ID NO:2 Amino acid sequence of mature CD59 (signal peptide removed).SEQ ID NO:3 Amino acid sequence of mature CD59 with glycated K41.SEQ ID NO:4 Amino acid sequence of mature CD59 glycated at: K14, K30,K38, K41, K65, K66, and K85.SEQ ID NO:5 TKAGLQVYNKCWKFEHCNFNDVTTRL with deuterated-(d₈)-V₍₃₅₎ andd₈-F₍₄₇₎.SEQ ID NO:6 AGLQVYNK with deuterated-(d₈)-V₍₃₅₎.SEQ ID NO:7 FEHCNFNDVTTR with deuterated-d₈-F₍₄₇₎.

SEQ ID NO:8 AGLQVYNK. SEQ ID NO:9 FEHCNFNDVTTR. SEQ ID NO:10CWKFEHCNFNDVTTR. SEQ ID NO:11 TKAGLQVYNKCWKFEHCNFNDVTTRL. SEQ ID NO:12AGLQV. SEQ ID NO:13 QVYNK. SEQ ID NO:14 AGLQVYNKCWKFEHCNFNDVTTRL. SEQ IDNO:15 AGLQVYNKCWKFEHCNFNDVTTR.

SEQ ID NO:16 AGLQVYNKCWKFEHCNFNDVTTRL with deuterated (d₈)-V₍₃₅₎ andd₈-F₍₄₇₎.SEQ ID NO:17 AGLQVYNKCWKFEHCNFNDVTTR with deuterated (d₈)-V₍₃₅₎ andd₈-F₍₄₇₎.

SEQ ID NO:18 CWKFEHCNFNDVTTRL.

SEQ ID NO:19 CWKFEHCNFNDVTTR with deuterated d₈-F₍₄₇₎.SEQ ID NO:20 CWKFEHCNFNDVTTRL with deuterated d₈-F₍₄₇₎.SEQ ID NO:21 AGLQVYNKCWKFEHCNFNDVTTR with a detectable label at V₍₃₅₎and F₍₄₇₎.SEQ ID NO:22 AGLQVYNK with a detectable label at V₍₃₅₎.SEQ ID NO:23 CWKFEHCNFNDVTTR with a detectable label at F₍₄₇₎.SEQ ID NO:24 FEHCNFNDVTTR with a detectable label at F₍₄₇₎.SEQ ID NO:25 TKAGLQVYNKCWKFEHCNFNDVTTR with deuterated-(d₈)-V₍₃₅₎ andd₈-F₍₄₇₎.

DETAILED DESCRIPTION

The invention disclosed herein describes novel methods and compositionsfor detecting and measuring glycated CD59 levels in samples includingbiological samples. The methods of the invention for analysis ofglycated CD59 in biological samples facilitate analysis of diseases inwhich the amount of CD59 glycation differs from normal levels. The levelof glycation of CD59 is elevated in diabetes. The methods of theinvention can be used to determine the onset, progression, and/orregression of diabetes or other diseases by using the methods to monitorlevels of glycated CD59 in a subject. The invention, in part, relates tothe comparison of the relative abundance of polypeptide products of theenzymatic digestion of glycated CD59, non-glycated CD59, and glycatedand non-glycated CD59 fragments as a determination of the level ofglycated CD59 in a biological sample.

The inventor generated an antibody that recognizes the glycated form ofhuman CD59 but not the non-glycated form nor other glycated proteins.Human CD59 was purified from human red blood cells and then glycated invitro by exposure to glucose 0.5M for variable times. The specificity ofthe antibody then was documented by both Western blot analysis andELISA. The antibody recognizes purified human CD59 after, but notbefore, glycation and does not recognize other glycated proteins such asglycated albumin, which is routinely used as a standard for glycatedproteins.

The anti-glycated CD59 antibody was used to measure by ELISA thepresence of glycated CD59 in human urine, plasma, kidney, nerve, andsaliva. For each sample type, an ELISA using an antibody against totalCD59 was also applied to the samples and the results expressed as theratio of glycated-CD59/Total CD59 (i.e. the relative amount of glycatedCD59 in each urine sample). The results indicated that glycated CD59 wasfound in human urine, plasma, kidney tissue, nerve tissue, and salivaand that the levels determined correlated well with the levels ofglycated hemoglobin (HbA1C) in blood, the current clinical standard forassessment of glycemic exposure in diabetic patients.

In addition, in contrast to markers of glycation such as hemoglobin,glycation of CD59 is believed to be involved in the pathogenesis of thevascular complications of diabetes. Accordingly, clinical evaluation ofglycated CD59 in urine or other body fluid is a direct measure forvascular damage induced by glycation. Without wishing to be bound by anyparticular theory, it is believed that patients with abnormally highlevels of glycated CD59 (e.g. K41-glycated CD59) in urine or other bodyfluid will either have or will be more prone to develop vascularcomplications of diabetes.

The inventor has now discovered novel methods, including massspectrometry methods, which can be used to assess the level of glycatedCD59 these samples from a subject. The methods, products, and kits ofthe invention are based, in part, on the novel discovery that therelative abundance of polypeptide products of enzymatic digestion ofglycated CD59, non-glycated CD59, and glycated and non-glycated CD59fragments can be compared and can provide a determination of the levelof glycated CD59 in a biological sample.

As used herein, CD59 (also known as membrane inhibitor of reactive lysis[MIRL], protectin, HRF20 and H19) and glycated CD59 are polypeptideshaving the amino acid sequence identity of Accession No. M95708 (Davies,A., et al., Journal J. Exp. Med. 170 (3), 637-654 (1989)). A nucleicacid sequence encoding CD59 also is provided by Davis, A, et al. A CD59sequence is provided herein as SEQ ID NO:1, which representsnon-glycated CD59. The sequence of non-glycated CD59 that is present inmature form in cells and tissues is set forth as SEQ ID NO:2. Thesequence of mature CD59 that is glycated at K41 is set forth as SEQ IDNO:3. The sequence of mature CD59 that is glycated at K14, K30, K38,K41, K65, K66, and K85 is set forth as SEQ ID NO:4.

As used herein, “glycated CD59” means CD59 that has been glycated. Insome embodiments, glycated CD59 is CD59 that has been glycated at theamino acid residue that corresponds to the amino acid residue number 41of full-length mature CD59, which is set forth herein as SEQ ID NO:2.The residue in position 41 of full-length mature CD59 is a lysine, andthis lysine in the full length and the residue that corresponds to thisposition in fragments is referred to herein as “K41”. CD59 in which theK41 residue is glycated is referred to herein as K41-glycated CD59. Insome embodiments, a glycated lysine residue is a glycocytol-lysineresidue. Thus, a glycated CD59 or fragment thereof may be glycated bythe inclusion of a glycocytol-lysine residue. In certain embodiments, alysine residue of CD59 or a fragment thereof may be glycated bycontacting the CD59 or fragment thereof with glycating sugars (e.g.glucose, ribose, or glycose-6-phosphate).

It is known that the CD59 polypeptide sequence includes a 25 amino acidsignal peptide that is cleaved when CD59 is produced, thus forming themature CD59 protein sequence. As would be understood by one of ordinaryskill in the art, CD59 in a sample obtained from a subject would be CD59from which the signal peptide has been cleaved. The sequence of the CD59polypeptide prior to removal of the signal peptide is provided herein asSEQ ID NO:1 and the amino acid sequence of mature CD59 polypeptide isset forth herein as SEQ ID NO:2. As used herein, the terms “peptide” and“polypeptide” are used to interchangeably.

Glycation of CD59, including, but not limited to K41 glycation of CD59,is correlated to abnormal blood sugar levels, and glycation of CD59interferes with the normal activity of CD59. CD59 functions normally bybinding to the terminal components of the membrane attack complex ofcomplement (MAC), thereby interfering with membrane insertion andpolymerization of the C9 component of complement. Glycation at the K41of CD59 interferes with CD59's ability to prevent the assembly of theMAC. While not wishing to be bound by any theory it is believed that, asa result of glycation of CD59, the MAC is permitted to be activated andleads to the development of proliferative chronic diabeticcomplications. Indeed, the present inventor has shown that the membraneattack complex stimulates proliferation of fibroblasts, smooth muscle,mesangial and other cells, in part by releasing growth factors such asFGF and PDGF from MAC-targeted endothelium. The MAC also inducesincreased synthesis of extracellular matrix proteins by mesangial cells.Thus, increased MAC deposition in diabetic tissues is believed to inducegrowth factor release from endothelium, which stimulates cellproliferation in the vascular wall and contributes to the expansion ofthe extracellular matrix and to the glomerulosclerosis thatcharacterizes diabetic nephropathy.

The invention also involves fragments of the foregoing proteins. Afragment of CD59 comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 or more contiguous amino acids of CD59having a consecutive sequence found in CD59 or a modified CD59 sequenceas described herein. As used herein, the term “CD59 peptide” includesfull-length CD59 and peptides that have an amino acid sequence thatcorresponds to a portion or fragment of full-length CD59. As usedherein, the term “portion” means part of the whole. In some embodiments,a fragment includes K41, which may or may not be glycated K41.Polypeptide fragments of the invention also include fragments of CD59generated by enzymatic digestion of CD59. In some embodiments, theenzymatic digestion is trypsin digestion. Fragments of CD59 moleculesinclude fragments of labeled CD59₍₂₉₋₅₄₎ peptide, includingdeuterated-(d₈)-V₍₃₅₎ and d₈-F₍₄₇₎ CD59₍₂₉₋₅₄₎ peptideTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:5), such as the fragmentsAGLQV_(d8)YNK (SEQ ID NO:6), CWK, and FEHCNF_(d8)NDVTTR (SEQ ID NO:7).The non-deuterated CD59₍₂₉₋₅₄₎ peptide (TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQID NO:11) and enzymatic cleavage fragments thereof are also peptides ofthe invention.

As used herein, the term “native peptide” means a peptide that isunlabeled and the term “labeled peptide” means a peptide that includes alabel. Thus, any native polypeptide of the invention may be referred toas a labeled polypeptide if it includes the same amino acid sequence asthe native polypeptide and also includes one or more labels. As will beunderstood by those of ordinary skill in the art, the nomenclature usedherein for the fragments of CD59 peptide and positions of labels on CD59peptide and fragments thereof, correspond to the residue numbers in themature CD59 molecule. For example, the residues indicated as 29-54 inthe name of the CD59₍₂₉₋₅₄₎ peptide correspond to residues 29-54 in themature CD59 peptide and detectable labels at V₍₃₅₎ and F₍₄₇₎ are on theresidues that correspond to V₃₅ and F₄₇ of mature CD59 peptide.

Additional fragments of the invention include enzyme digestion productsof glycated and non-glycated CD59. In some embodiments, the enzymedigestion is a trypsin digestion. Fragments that result from trypsindigestion of non-glycated CD59 include at least AGLQVYNK (SEQ ID NO:8),CWK, and FEHCNFNDVTTR (SEQ ID NO:9). Fragments that result from thetrypsin digestion of glycated CD59 include at least AGLQVYNK (SEQ IDNO:8) and CWKFEHCNFNDVTTR (SEQ ID NO:10). The digestion products thatresult from trypsin digestion of glycated CD59 differ from the digestionproducts that result from trypsin digestion of non-glycated CD59, thuspermitting the comparison of the resulting digestion products as arelative measure of the amounts of glycated and non-glycated CD59 in adigested sample.

In some embodiments, a CD59 polypeptide of the invention includes adetectable label. In some embodiments, the detectable label is selectedfrom the group consisting of a fluorescent label, an enzyme label (e.g.biotinylation, etc.), a radioactive label (e.g ¹⁴C, ³H, etc.), a nuclearmagnetic resonance active label, a luminescent label (e.g fluoresceinisothiocyanate, etc.), and a chromophore label. In some embodiments, thelabel is a label used for mass spectrometry methods. In someembodiments, stable isotopes may be used as labels, including, but notlimited to: carbon 13 (¹³C), protium (¹H), deuterium (²H), nitrogen 15(¹⁵N), oxygen 17 (¹⁷O), oxygen 18 (¹⁸O), sulfur 34 (³⁴S), sulfur 33(³³S), chlorine 37 (³⁷Cl), bromine 81 (⁸¹Br), and beryllium 10 (¹⁰Be).In some embodiments, a CD59 polypeptide of the invention is a deuteratedpolypeptide. Additional labels suitable for mass spectrometry and otherdetection methods useful in the methods and kits of the invention willbe known to those of skill in the art. In the methods and kits of theinvention, a label may be attached to a peptide, e.g. covalentlyattached, and/or may be integrated into the peptide. As used herein theterm “integrated into” means that the label may be a part of thepeptide—e.g. a peptide may be synthesized with a deuterated amino acid,thus the label is included in the polypeptide and is a part of thepolypeptide.

According to one aspect of the invention, the detectable label may beattached to or integrated into a CD59 polypeptide or fragments thereof,such that upon enzymatic digestion, the label remains attached to or asan integral part of a digestion product of the CD59 polypeptide or CD59polypeptide fragments. For example, TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ IDNO:11) may be labeled with one or more detectable labels such thatfollowing trypsin digestion, the detectable label remains on one or moreof the fragments generated in the digest. For example, a label may beattached to SEQ ID NO:11 such that following digestion with trypsin, thelabel will remain on a digestion product of the CD59 polypeptidefragment. Thus, if SEQ ID NO:11 was originally labeled with a detectablelabel on V₍₃₅₎, after trypsin digestion of a sample containing thelabeled SEQ ID NO:11, the label will be on the digestion product:FEHCNFNDVTTR (SEQ ID NO:9).

In some embodiments of the invention, a sample is spiked with a labeledCD59 polypeptide or spiked with labeled CD59 polypeptide fragments. Whenadded to a sample, the detectably labeled CD59 or CD59 fragments serveas an internal standard in the sample. As used herein, the term “tospike” means to add the internal standard to a sample. The spiking of asample with a known amount of a CD59 polypeptide or fragment thereofallows detection and quantitation of the digestion products of the spikeCD59 polypeptide (e.g. the detectibly labeled CD59 polypeptide) and thedetermination of the relative amounts of other digestion products in thesample. As used herein, the term “internal standard” means a knownamount of a CD59 polypeptide or fragment thereof. In some embodiments,the internal standard is a detectably labeled CD59 polypeptide ordetectably labeled fragment thereof. The presence of a detectable labelon a fragment of a digested sample allows the quantities of labeled andunlabeled fragments to be determined and compared allowing thequantitation of the amount of glycated and/or non-glycated CD59 in thesample.

As provided herein, CD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQID NO:5) with deuterated-(d₈)-V₍₃₅₎ and d₈-F₍₄₇₎ is an example of alabeled CD59 fragment that can be added as an internal standard to asample in some embodiments of the invention. It will be understood bythose of ordinary skill in the art that in some embodiments of theinvention the label on the CD59 polypeptide may be in a differentposition on the polypeptide (e.g. on a residue other than V₍₃₅₎ orF₍₄₇₎). In addition, the label may be a label other than a deuteratedlabel. Thus, for use with mass spectrometry detection methods andrelated kits of the invention, a deuterated label or other massspectrometry-appropriate label may be used. One of ordinary skill willunderstand that for use with other detection methods such aschromatography, ELISA, or electrophoresis, other (e.g.method-appropriate) detectable labels may be used.

Additional CD59 polypeptide fragments are useful in the methods and kitsof the invention. For example a deuterated or non-deuterated polypeptidethat includes fewer or more amino acids than TKAGLQVYNKCWKFEHCNFNDVTTRL(SEQ ID NO:11) can be used in the methods of the invention. For example,deuterated or non-deuterated polypeptides with an amino acid sequenceAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:14) or AGLQVYNKCWKFEHCNFNDVTTR (SEQID NO:15) can be used in the methods of the invention, as can apolypeptide that is 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid residuesshorter than the sequence set forth as SEQ ID NO:11, as long as thefragment permits quantitation of glycated CD59 using a method providedherein.

In one embodiment, the fragment is AGLQVYNKCWKFEHCNFNDVTTR (SEQ IDNO:15). This fragment, if glycated at K41, will be digested by trypsininto AGLQVYNK (SEQ ID NO:8) and CWKFEHCNFNDVTTR (SEQ ID NO:10) but ifnon-glycated the fragment will be digested into: AGLQVYNK (SEQ ID NO:8),CWK, and FEHCNFNDVTTR (SEQ ID NO:9). In some embodiments of theinvention, the sequence: AGLQVYNKCWKFEHCNFNDVTTR (SEQ ID NO:15) includesone or more labels such that when the fragment is digested by trypsin, alabel will be on fragments generated by the digestion. For example,AGLQVYNKCWKFEHCNFNDVTTR (SEQ ID NO:15) may include a label at V₍₃₅₎ andF₍₄₇₎ residues as in AGLQV_(label)YNKCWKFEHCNF_(label)NDVTTR (SEQ IDNO:21). In some embodiments the label is a deuterated label. Afterdigestion of the glycated labeled polypeptide set forth as SEQ ID NO:15,the resulting fragments will be AGLQV_(label)YNK (SEQ ID NO:22) andCWKFEHCNF_(label)NDVTTR (SEQ ID NO:23). If the labeled polypeptide setforth as SEQ ID NO:15 is non-glycated the resulting digest fragmentswill be AGLQV_(label)YNK (SEQ ID NO:22), CWK, and FEHCNF_(label)NDVTTR(SEQ ID NO:24). The production of different digestion products, and insome embodiments, differentially labeled digestion products allowsidentification of the fragments generated by trypsin digestion andidentification of the presence of glycated and non-glycated CD59 in thedigested sample. One of ordinary skill will understand that the residueon which a label is attached can vary from the example provided above.The specific residue that is labeled is selected based on it enablingone to differentiate the digestion products from glycated versusnon-glycated CD59.

In some embodiments, a fragment of CD59 that is useful in the methods ofdetermining the amount of glycated CD59 in a sample can be a fragment ofCD59 larger than the fragment set forth as SEQ ID NO:11. In someembodiments of the invention, the spike polypeptide can be full-lengthnon-glycated CD59 or can be non-glycated CD59 that is one or more aminoacid shorter than full-length CD59. In some embodiments, the fragment ofCD59 can be labeled (e.g. deuterated in the manner of SEQ ID NO:5 orotherwise labeled). The fragments can be used to determine the amount ofglycated CD59 in a sample with a mass spectrometry method or otherdetermining method provided herein.

According to some aspects of the invention, isolated CD59 polypeptidesare provided. By “isolated”, it is meant present in sufficient quantityto permit its identification or use according to the proceduresdescribed herein. Isolated includes (1) synthesized, (2) selectivelyproduced by expression cloning or (3) purified as by mass spectrometry,immunoprecipitation, chromatography, or electrophoresis. Because anisolated material may be admixed with a carrier in a preparation, suchas, for example, for adding to a sample or for analysis, the isolatedmaterial may comprise only a small percentage by weight of thepreparation.

The term “isolated polypeptide” may also mean a polypeptide that is notin association with amino acids with which it is naturally found. Forexample, an isolated AGLQVYNK (SEQ ID NO:8) polypeptide is a polypeptidethat does not include the amino acids that correspond to the amino acidsadjacent to this portion of full-length, mature CD59 polypeptide. Thus,for isolated SEQ ID NO:8, the amino acids beginning at (and extendingoutward from) the amino acids K and C that are adjacent to the SEQ IDNO:8 portion of the sequence in full-length, mature CD59, are notpresent in isolated SEQ ID NO:8. Thus, the term “isolated” means thepolypeptide is separated from (free of) the adjacent amino acids thatwould be present in mature, full-length CD59 polypeptide.

According to one aspect of the invention, isolated glycated CD59polypeptides and fragments are provided. An isolated glycated CD59polypeptide or fragment thereof may include CD59 polypeptide or fragmentthereof (1) selectively produced by expression cloning and glycation or(2) purified as by mass spectrometry, immunoprecipitation,chromatography, or electrophoresis. Because an isolated material may beadmixed with a carrier or solvent in a preparation, the isolatedmaterial may comprise only a small percentage by weight of thepreparation. The material is none-the-less isolated in that it has beenseparated from the substances with which it typically is associated.According to the present invention, the isolated glycated CD59represents at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%of the CD59 in the composition (i.e. percentage of total CD59 bothglycated and nonglycated CD59).

According to another aspect of the invention, pure glycated CD59 isprovided. In some embodiments, the pure glycated CD59 is K41-glycatedCD59. Isolated proteins or polypeptides may, but need not be, pure. Theterm “pure” means the proteins or polypeptides are essentially free ofother substances with which they may be found in nature or in in vivosystems to an extent practical and appropriate for their intended use.Pure polypeptides may be produced by techniques well-known in the art.As used herein, “pure” glycated CD59 means at least 95% of the totalCD59 is glycated CD59.

As used herein, the term “glycated CD59” polypeptide includes matureCD59 polypeptide with one or more glycated lysine (K) residues. In someembodiments, the glycated lysine residue of CD59 is residue K41 ofmature CD59. One of ordinary skill in the art will understand that afragment of CD59 can be compared to mature full-length CD59, and thepresence of a residue in that fragment is said to “correspond” to theresidue of mature CD59. As used herein therefore, residue positions forlysines are identified as they occur in mature CD59, whether thatresidue is part of mature CD59 or part of a fragment or modifiedfragment. Thus, K41 maintains that designation in mature CD59 orfragments thereof. In some embodiments, the glycated lysine residue in afragment of CD59 is K41. In certain embodiments of the invention, theglycated residue of CD59 or a fragment thereof is or corresponds to K14,K30, K38, K65, K66, or K85 of mature CD59 polypeptide. In someembodiments, more than one K residue is glycated.

The methods of the invention include mass spectrometry analysis. Massspectrometry is an analytical method that is used for used forquantitative and qualitative analysis of the samples and materials(e.g., biological samples). In general, a mass spectrometry system usesan ion source to produce electrically charged particles (e.g., molecularor polyatomic ions) from the material to be analyzed. The electricallycharged particles are introduced to the mass spectrometer and separatedby a mass analyzer based on the particles' respective mass-to-chargeratios. The abundance of the separated electrically charged particlesare detected and a mass spectrum of the material is produced. The massspectrum provides information about the mass-to-charge ratio of aparticular compound in a mixture sample and, in some cases, molecularstructure of that component in the mixture. In some embodiments of theinvention, mass spectrometry is isotope ratio mass spectrometry.

Mass spectrometers convert components of a sample into rapidly movinggaseous ions and separate them on the basis of their mass-to-chargeratios. Generally, mass spectrometers include an ionization means and amass analyzer. A number of different types of mass analyzers are knownin the art. These include, but are not limited to, magnetic sectoranalyzers, electrostatic analyzers, quadrapoles, ion traps,time-of-flight mass analyzers, and fourier transform analyzers. Inaddition, two or more mass analyzers may be coupled (MS/MS) first toseparate precursor ions, then to separate and measure gas phase fragmentions.

Mass spectrometers may also include one of a number of differentionization methods. These include, but are not limited to, gas phaseionization sources such as electron impact, chemical ionization, andfield ionization, as well as desorption sources, such as fielddesorption, fast atom bombardment, matrix assisted laserdesorption/ionization, and surface enhanced laser desorption/ionization.In addition, mass spectrometers may be coupled to separation means suchas gas chromatography (GC) and high performance liquid chromatography(HPLC).

In gas-chromatography mass-spectrometry (GC/MS), capillary columns froma gas chromatograph are coupled directly to the mass spectrometer,optionally using a jet separator. In such an application, the gaschromatography (GC) column separates sample components from the samplegas mixture and the separated components are ionized and chemicallyanalyzed in the mass spectrometer. Thus, in GC/MS analysis, a mixture ofcompounds to be analyzed is first injected into the gas chromatographand is vaporized in a heated chamber. The gas mixture then goes througha GC column, where the compounds are separated as they interact with thecolumn. A gas chromatogram for the sample is generated showing peaks ofthe separated compounds from sample, and the separated compoundsimmediately enter the mass spectrometer for MS analysis.

There are three general elements or components that make up a massspectrometry system including: the ionizer, the ion analyzer, and thedetector. One of ordinary skill in the art will understand how to applythe MS methods and embodiments of the invention utilizing MS systemsthat include combinations of the MS components and elements describedherein, as well as other art-known MS methods and procedures.

Samples must be in gaseous form for MS analysis. Thus, in someembodiments, a sample may be vaporized by heating. After it is vaporizedthe sample passes through the electron ionization component of the MSsystem. One of ordinary skill will recognize that a number of ionizationmethods are known in the art that can be used in the methods of theinvention. For example, electron ionization is useful in conjunctionwith GS/MS. Other methods of ionization that are useful in the methodsof the invention include, but are not limited to: ElectrosprayIonization (ESI), Matrix-Assisted Laser Desorption/Ionization MassSpectrometry (MALDI-MS), atmospheric pressure chemical ionization(APCI), Fast Atom Bombardment (FAB), chemical ionization (CI) andInductively Coupled Plasma (ICP) ionization.

After ionization, the next stage of MS is mass analysis. This occurs inthe mass analyzer of the MS. The mass analyzer separates ions within aselected range of mass-to-charge (m/z) ratios. The basis for thisseparation generally is through the use of magnetic fields, electricfields, or measurement of time it takes for an ion to travel a specifieddistance. Known methods for mass analysis in MS methods include, but arenot limited to: double focusing magnetic sector mass analysis, tandemmass spectrometry, Quadrupole mass analysis, Quadrupole ion trap massanalysis, Fourier-Transform Mass Spectrometry (FTMS), and Time-of-Flight(TOF). The final element of MS analysis is ion detection. Ion detectionmeans known in the art include, but are not limited to: faraday cupdetectors, electron multiplier detectors, and photomultiplier conversiondynode detectors.

One of ordinary skill in the art will understand how methods and kits ofthe invention may be optimized using combinations of the MS components,elements, and method described herein as well as other art-known MSapparatus and apparatus.

In one embodiment of the invention, the typsin digestion products of asample may be measured directly by mass spectrometry. In anotherembodiment, typsin digestion products of a sample may be partiallypurified, or optionally isolated, prior to mass spectral analysis. Abiological sample, e.g. a sample from a subject, may be prepared formass spectrometry analysis using standard procedures known in the art.An example of sample preparation methods useful in the methods of theinvention is provided in the Example section.

For mass spectrometry analysis of the invention, an internal standard ofa known amount of a CD59 peptide is added to the sample prior totrypsinization. In some embodiments, the internal standard is theCD59₍₂₉₋₅₄₎ peptide TKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:5),which includes deuterated-(d₈)-V₍₃₅₎ and d₈-F₍₄₇₎, is added to eachsample prior to trypsinization. As used herein, the term “spike” meansto add the internal standard to a sample. For example, a spiked sampleis a sample to which an internal standard of CD59₍₂₉₋₅₄₎ peptideTKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:5) has been added. Inimportant embodiments, the amount of the internal standard added is aknown amount. The sample with the internal standard added (spikedsample) is then the sample is trypsinized to digest the spiked sample.Trypsin digestion can be done under conditions that allow the digestionof the spiked sample to generate fragments of the polypeptides in thesample. For example, the enzymatic digestion is carried out underconditions that allow the glycated and non-glycated CD59 and theinternal standard CD59₍₂₉₋₅₄₎ peptideTKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL; SEQ ID NO:5) to be digested intodetectable fragments. In some embodiments, the digestion may bedigestion of the full-length starting polypeptide to completion. In theembodiments of the invention, the conditions for the enzymatic digestionof the spiked sample are sufficient for the glycated, non-glycated, andCD59₍₂₉₋₅₄₎ peptide to be digested into the fragments described herein.Digestion conditions may include a temperature of up to about 20° C.,21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C.,30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C.,39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., or more and alength of digestion from up to about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, ormore minutes. The pH of the digest reaction may be in a rangeappropriate for the enzyme, e.g. trypsin. For example, for a trypsindigestion, the pH may optimally range from about 7 to about 9 includingany pH level in between 7 and 9. In one embodiment, the temperature ofthe digestion is about 37° C. and the length of time of the digestion isabout 30 minutes.

One of ordinary skill in the art will recognize that the optimaltemperature, pH, and length of time of the trypsin digestion can bevaried based on factors such as enzyme activity, enzyme amount, etc. Inaddition, the length of time of the digestion may also be varieddepending on the temperature of the digestion and vice versa. One ofskill in the art can easily optimize the time, temperature,concentration, etc. of the trypsin digestion conditions for use in themethods and kits of the invention. One example of conditions that can beused in the methods of the invention, although not intended to belimiting, is a 30 minute digestion at 37° C.

In some embodiments of the invention, the enzyme used in the digestionmethods to determine the level of glycated CD59 is trypsin. Trypsin maybe naturally obtained trypsin, or may be artificial trypsin, e.g.recombinant trypsin. The source of trypsin useful in the methods of theinvention, include, but is not limited to, bovine, porcine, human, fish,etc. The trypsin of may be free or immobilized trypsin. The trypsin maybe treated with an agent to remove chymotrypsin activity. For examplethe trypsin may be treated with diphenyl carbamyl chloride (DPCC) orwith L-(tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK) to reducethe activity level of chymotrypsin. Those of ordinary skill willunderstand that trypsin is known by other names and can be obtained invarious levels of purity, grade, and forms. For example the trypsin maybe highly purified for use in mass spec. The trypsin may be modifiedtrypsin. An example of a modified trypsin, though not intended to belimiting is trypsin modified by reductive methylation to increasestability.

Trypsinization of the urine or plasma sample (or other biological sampleor control) with the added internal standard d₈-CD59₍₂₉₋₅₄₎ peptideyields the different polypeptide digestion products of CD59 depending onwhether the CD59 is glycated or non-glycated CD59. If the CD59 isnon-glycated CD59, the tryptic digestion methods of the invention yieldsthe following peptides: AGLQVYNK (SEQ ID NO:8), CWK, and FEHCNFNDVTTR(SEQ ID NO:9). Trypsin digestion of CD59₍₂₉₋₅₄₎ peptideTKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:5) yields AGLQV_(d8)YNK(SEQ ID NO:6), CWK, and FEHCNF_(d8)NDVTTR (SEQ ID NO:7). In contrast:trypsin digestion of glycated CD59 yielded only two peptides becauseglycated CD59 is not cleaved at the glycated K41 site: AGLQVYNK (SEQ IDNO:8) and CWKFEHCNFNDVTTR (SEQ ID NO:10).

Isotopically labeled and natural peptides ionize in an identical manner,thus the ionization of the internal standard peptides [AGLQV_(d8)YNK(SEQ ID NO:6) and FEHCNF_(d8)NDVTTR (SEQ ID NO:7)] is the same as theionization of the peptides derived from the subject's CD59 [AGLQVYNK(SEQ ID NO:8) and FEHCNFNDVTTR (SEQ ID NO:9)]. Thus, peptides derivedfrom either the internal standard or the subject's CD59 have identicalcharge but a different mass of eight (8) units that allows theirunambiguous identification and quantitation provided that the amount ofthe labeled CD59₍₂₉₋₅₄₎ peptide added to the sample was known. The massdifference of 8 is the result of the presence of V_(d8) in AGLQV_(d8)YNK(SEQ ID NO:6) and of F_(d8) in FEHCNF_(d8)NDVTTR (SEQ ID NO:7).

The AGLQVYNK (SEQ ID NO:8) peptide is generated by the trypsin digestionof both glycated and non-glycated CD59. Thus, the quantitative estimateof AGLQVYNK (SEQ ID NO:8) represents total CD59 in the sample. Incontrast, the FEHCNFNDVTTR (SEQ ID NO:9) peptide is only generated bythe trypsin digestion of non-glycated CD59. Thus, the quantitativeestimate of FEHCNFNDVTTR (SEQ ID NO:9) represents non-glycated CD59 inthe sample. The difference between the amount of total CD59 and theamount of non-glycated CD59 is the amount of glycated CD59 in thesample. In some embodiments of the invention, a sample is a controlsample, e.g. a sample from a subject known to not have a diabeticdisorder or abnormal ability to metabolize glucose.

As detailed herein, the mass spectrometric methods of the invention maybe used for example to identify and quantitate CD59 protein (includingK41-nonglycated CD59) and/or glycated CD59 protein includingK41-glycated protein. Thus, in the method and products of the invention,the inclusion of a known amount of an internal standard (e.g.CD59₍₂₉₋₅₄₎ peptide TKAGLQV_(d8)YNKCWKFEHCNF_(d8)NDVTTRL (SEQ ID NO:5)in the sample to be tested permits the determination of the level ofglycated CD59 in the sample.

In other embodiments of the invention, the fragments of CD59 andglycated CD59 generated with enzymatic digestion, e.g. trypsindigestion, are determined using non-mass spectrometric methods. Forexample additional methods such as immunoassay, (e.g. ELISA, etc.) gelelectrophoresis, Western blot analysis, NMR, chromatography, etc. may beused to identify the fragments produced by the digestion of glycated andnon-glycated CD59. For example, as described above, if non-glycated CD59is digested by typsin the resultant peptides are: AGLQVYNK (SEQ IDNO:8), CWK, and FEHCNFNDVTTR (SEQ ID NO:9). In contrast: trypsindigestion of glycated CD59 yields only two peptides, AGLQVYNK (SEQ IDNO:8) and CWKFEHCNFNDVTTR (SEQ ID NO:10), because glycated CD59 is notcleaved and the glycated K41 site. Thus, AGLQVYNK (SEQ ID NO:8) isgenerated by the trypsin digestion of both glycated and non-glycatedCD59, so the quantitative estimate of AGLQVYNK (SEQ ID NO:8) in a samplerepresents total CD59 in the sample. In contrast, the FEHCNFNDVTTR (SEQID NO:9) peptide is only generated by the trypsin digestion ofnon-glycated CD59, therefore the level of FEHCNFNDVTTR (SEQ ID NO:9)present in a digested sample can be used as a quantitative measure ofthe amount of non-glycated CD59 in the sample. In some embodiments, thelevel of CD59 in a sample can be determined using the digest methods ofthe invention and a comparison of the relative amounts of CWK and SEQ IDNO:10 in a digested sample.

The difference between the level (amount) of total and the amount ofnon-glycated CD59 is the amount of glycated CD59 in the sample. As usedherein, the term “level” means amount. For example, the level ofglycated CD59 in a sample is the amount of glycated CD59 in the sample.The amount can be a relative amount, or absolute amount, depending onthe technique employed, as will be clear from context.

According to some aspects of the invention, agents that bindspecifically to peptides generated by the digestion of glycated,non-glycated CD59, or that bind to digestion products of a fragment ofCD59 [e.g. CD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ IDNO:11)] can be prepared and used to identify and quantitate the amountof glycated CD59 in a sample. As used herein, “binding specifically to”means capable of distinguishing the identified material from othermaterials sufficient for the purpose to which the invention relates. Forexample, “binding specifically to” AGLQVYNK (SEQ ID NO:8), CWK,FEHCNFNDVTTR (SEQ ID NO:9), or CWKFEHCNFNDVTTR (SEQ ID NO:10) means theability to bind to and distinguish AGLQVYNK (SEQ ID NO:8), CWK,FEHCNFNDVTTR (SEQ ID NO:9), or CWKFEHCNFNDVTTR (SEQ ID NO:10) from eachother and/or from other peptides and proteins.

Agents that bind to digestion products of glycated CD59, non-glycatedCD59, or digestion products of a fragment of CD59 [e.g. CD59₍₂₉₋₅₄₎peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)] include polypeptideagents. Such polypeptides include polyclonal and monoclonal antibodies,prepared according to conventional methodology.

In some embodiments, antibodies or antigen-binding fragments thereofthat specifically bind to a fragment generated by digestion of a CD59polypeptide can be used to assess the presence of glycated CD59polypeptides in a sample. For example, an antibody or antigen-bindingfragment thereof that can distinguish the fragments generated bydigestion of glycated CD59 from the fragments generated by digestion ofnon-glycated CD59 can be used to indicate the presence and/or level ofglycated CD59 in a sample.

Significantly, as is well known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology, Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)2 fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd Fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, W. R. (1986) The Experimental Foundations of ModernImmunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) EssentialImmunology, 7th Ed., Blackwell Scientific Publications, Oxford). In boththe heavy chain Fd fragment and the light chain of IgG immunoglobulins,there are four framework regions (FR1 through FR4) separatedrespectively by three complementarity determining regions (CDR1 throughCDR3). The CDRs, and in particular the CDR3 regions, and moreparticularly the heavy chain CDR3, are largely responsible for antibodyspecificity.

It is now well established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762 and 5,859,205.

Thus, for example, PCT International Publication Number WO 92/04381teaches the production and use of murine RSV antibodies in which atleast a portion of the murine FR regions have been replaced by FRregions of human origin. Such antibodies, including fragments of intactantibodies with antigen-binding ability, are often referred to as“chimeric” antibodies. Fully human monoclonal antibodies also can beprepared by immunizing mice transgenic for large portions of humanimmunoglobulin heavy and light chain loci. Following immunization ofthese mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)),monoclonal antibodies can be prepared according to standard hybridomatechnology. These monoclonal antibodies will have human immunoglobulinamino acid sequences and therefore will not provoke human anti-mouseantibody (HAMA) responses when administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)2, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)2 fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornonhuman sequences. The present invention also includes so-called singlechain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to digestion products of glycated CD59, non-glycatedCD59, or a fragment of CD59 (glycated or non-glycated) such asCD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11) molecules.Thus, in some embodiments, an antibody that specifically binds to afragment of glycated CD59 generated by enzyme digestion (e.g. trypsindigestion) and does not bind to fragments generated from a similardigestion of non-glycated CD59 can be used to determine relative amountsof glycated and non-glycated CD59 in the sample. Thus, using thedifferential digestion of glycated and non-glycated CD59 peptides intodistinct fragments allows the determination of the presence and/oramount of glycated CD59 in a sample. One of ordinary skill willrecognize that the different fragments generated by the digestion ofglycated versus non-glycated CD59 peptides allow the use of bindingpeptides (e.g. antibodies) that specifically bind to certain fragmentsto determine the presence and/or amount of glycated CD59 in a sample.

These binding polypeptides may be derived also from sources other thanantibody technology. For example, such polypeptide binding agents can beprovided by degenerate peptide libraries that can be readily prepared insolution, in immobilized form or as phage display libraries.Combinatorial libraries also can be synthesized of peptides containingone or more amino acids. Libraries further can be synthesized ofpeptoids and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind to adigestion product of glycated CD59, non-glycated CD59, or fragment ofglycated or non-glycated CD59 [e.g. CD59₍₂₉₋₅₄₎ peptideTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)] molecules. This process canbe repeated through several cycles of reselection of phage that bind toa digestion product of glycated CD59, non-glycated CD59, or fragment ofglycated or non-glycated CD59 [e.g. CD59₍₂₉₋₅₄₎ peptideTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)] molecules. Repeated roundslead to enrichment of phage bearing particular sequences. DNA sequencesanalysis can be conducted to identify the sequences of the expressedpolypeptides. The minimal linear portion of the sequence that binds to adigestion product of glycated CD59, non-glycated CD59, or fragment ofglycated or non-glycated CD59 [e.g. CD59₍₂₉₋₅₄₎ peptideTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)] molecules can be determined.One can repeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Yeasttwo-hybrid screening methods also may be used to identify polypeptidesthat bind to a digestion product of glycated CD59, non-glycated CD59, orfragment of glycated or non-glycated CD59 [e.g. CD59₍₂₉₋₅₄₎ peptideTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)] molecules. Thus, digestionproducts of glycated CD59, non-glycated CD59, or fragment of glycated ornon-glycated CD59 [e.g. CD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL(SEQ ID NO:11)] molecules can be used to screen peptide libraries,including phage display libraries, to identify and select peptidebinding partners of the digestion products of glycated CD59,non-glycated CD59, or fragments of glycated or non-glycated CD59 [e.g.CD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)]molecules.

As detailed herein, the foregoing antibodies and other binding moleculesmay be used for example to identify digestion products of glycated CD59,non-glycated CD59, or fragment of glycated or non-glycated CD59 [e.g.CD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)] moleculesand can be used to determine the amount of glycated CD59 in a sample.The antibodies may be coupled to specific diagnostic labeling agents forimaging of a digestion product of glycated CD59, non-glycated CD59, orfragment of glycated or non-glycated CD59 [e.g. CD59₍₂₉₋₅₄₎ peptideTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:11)] molecules. The antibodies mayalso be used for immunoprecipitation, immunoblotting digestion productsof glycated CD59, non-glycated CD59, or fragment of glycated ornon-glycated CD59 [e.g. CD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL(SEQ ID NO:11)] molecules, using standard methods known to those ofordinary skill in the art.

The invention provides methods and kits for the determination of thefragments produced in enzymatic digests of samples that are not spikedby allowing the quantitation of one or more of the fragments thatspecifically represent digestion products of either CD59 that isglycated or CD59 that is not glycated. For example, in some embodiments,the presence and/or level of CWK and AGLQVYNK (SEQ ID NO:8) can bedetermined. The identification of a lower level of CWK than AGLQVYNK(SEQ ID NO:8) in a sample indicates that glycated CD59 was present inthe sample. In some embodiments, the amount of a peptide in a digestedsample is quantified. The quantitation of one or more peptides in adigested sample provides a determination of the initial amount ofglycated and/or non-glycated CD59 in the sample.

The invention also involves a variety of assays based upon detectinglevels of glycated CD59 in subjects. The assays include (1)characterizing the impact of blood sugar levels on glycation levels in asubject; (2) evaluating a treatment for regulating blood sugar levels ina subject; (3) selecting a treatment for regulating blood sugar levelsin a subject; and (4) determining progression, progression or onset of acondition characterized by abnormal levels of glycated protein in asubject. Thus, subjects can be characterized, treatment regimens can bemonitored, treatments can be selected and diseases can be betterunderstood using the assays of the present invention. For example, theinvention provides in one aspect a method for measuring the level ofglycated CD59 in a subject, which is a direct indicator of the level ofthe subject's glycemic control. The impact of blood sugar levels orglycation levels thus can be measured due to the positive correlationbetween the level of circulating blood glucose and the amount ofglycation of endogenous CD59. The level of glycated CD59 thus correlateswith the level of glycemic control in the subject. Relatively low levelsof glycated CD59 reflect well-controlled circulating blood sugar levelsand selectively high levels of glycated CD59 reflect poorly controlledglycemic levels. CD59 is present in body fluids of subjects with andwithout diabetes. For example, the concentration of CD59 in urine isgenerally in a range from about 4 to 8 μg/ml of urine. In a subject withuncontrolled diabetes, the amount of the CD59 that is glycated CD59(e.g. K41-glycated CD59) in a bodily fluid is about 50% to 60% of theCD59 present in the sample. Thus, a percentage of CD59 in a sample canbe used as a determination of the glycemic status of the subject, andthe percentage of glycated CD59 reflects the subject's regulation ofsugar levels, e.g. the subject's level of glycemic control. A higherpercentage in a sample from a subject than that found in a normal sampleindicates that the subject has reduced glycemic control compared to thelevel of glycemic control in the subject providing the normal sample.

The assays described herein are carried out on samples. In someembodiments, a sample is a biological sample obtained from a subject. Insome embodiments, a sample can be synthetic or (e.g. laboratoryprepared) and not obtained from a subject. As used herein, a subject isa human, non-human primate, cow, horse, pig, dog, cat, or rodent. In allembodiments, human subjects are preferred.

As used herein, a “biological sample” encompasses a variety of sampletypes obtained from an individual through invasive or non-invasiveapproaches (e.g., urine collection, blood drawing, needle aspiration,and other procedures). The definition also includes samples that havebeen manipulated in any way after their procurement (through invasive ornon-invasive approaches), such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” includes, but is notlimited to, any body tissue or body fluid sample obtained from asubject. Body fluids include: urine, blood, saliva, lacrimal fluid,synovial fluid, cerebrospinal fluid, sweat, pulmonary secretions(sputum), seminal fluid, and feces. Preferred are body fluids, forexample, lymph, saliva, blood, urine, and the like.

Particularly important subjects to which the present invention can beapplied are diabetic subjects and subjects at risk for diabetes or otherglucose metabolism abnormality—e.g. a subject with reduced glycemiccontrol (ability to regulate sugar levels) compared to a normal subject.

The term “diabetic” as used herein, means an individual who, at the timethe sample is taken, has a primary deficiency of insulin and/or anabnormal (e.g. reduced) ability to metabolize glucose, e.g. impairedglucose tolerance versus a normal subject. The term diabetic includes,but is not limited to, individuals with juvenile diabetes (Type 1diabetes), adult-onset diabetes (Type 2 diabetes), gestational diabetes,and any other conditions of insulin deficiency or reduction in theability to metabolize glucose. A subject who is an uncontrolled or notfully controlled diabetic has reduced glycemic control as compared to asubject who is not diabetic or is a controlled diabetic subject. Theterm “diabetic” is a term of art, known and understood by thosepracticing in the medical profession, a formal definition of which canbe found in Harrison's Principles of Medicine (Harrisons, Vol 14,Principles of Internal Medicine, Eds. Fauci, A. S., E. Braunwald, K. J.Isselbacher, J. D. Wilson, J. B. Martin, D. L. Kasper, S. L. Hauser, D.L. Longo, McGraw-Hill, New York, 1999).

The assays described herein involve measuring levels of glycated CD59.Levels of glycated CD59 can be determined in a number of ways whencarrying out the various methods of the invention. In one particularlyimportant measurement, the level of glycated CD59 is measured inrelation to nonglycated CD59. Thus, the measurement is a relativemeasure, which can be expressed, for example, as a percentage of totalCD59. Another measurement of the level of glycated CD59 is a measurementof absolute levels of glycated CD59. This could be expressed, forexample, in terms of grams per liter of body fluid. Another measurementof the level of glycated CD59 is a measurement of the change in thelevel of glycated CD59 over time. This may be expressed in an absoluteamount or may be expressed in terms of a percentage increase or decreaseover time.

Importantly, levels of glycated CD59 are advantageously compared tocontrols according to the invention. The control may be a predeterminedvalue, which can take a variety of forms. It can be a single cut-offvalue, such as a median or mean. It can be established based uponcomparative groups, such as in groups having normal amounts ofcirculating insulin and groups having abnormal amounts of circulatinginsulin. Another example of comparative groups would be groups having aparticular disease, condition or symptoms and groups without thedisease, condition or symptoms. Another comparative group would be agroup with a family history of a condition and a group without such afamily history. The predetermined value can be arranged, for example,where a tested population is divided equally (or unequally) into groups,such as a low-risk group, a medium-risk group and a high-risk group orinto quadrants or quintiles, the lowest quadrant or quintile beingindividuals with the lowest risk or amounts of glycated protein and thehighest quadrant or quintile being individuals with the highest risk oramounts of glycated protein.

The predetermined value, of a course, will depend upon the particularpopulation selected. For example, an apparently healthy population willhave a different ‘normal’ range than will a population that is known tohave a condition related to abnormal protein glycation. Accordingly, thepredetermined value selected may take into account the category in whichan individual falls. Appropriate ranges and categories can be selectedwith no more than routine experimentation by those of ordinary skill inthe art. By abnormally high it is meant high relative to a selectedcontrol. Typically the control will be based on apparently healthynormal individuals in an appropriate age bracket.

In measuring the relative amount of glycated CD59 to nonglycated CD59,those of ordinary skill in the art will appreciate that the relativeamount may be determined by measuring either the relative amount ofglycated CD59 or the relative amount of nonglycated CD59. In otherwords, if 90% of an individual's CD59 is nonglycated CD59, then 10% ofthe individual's CD59 will be glycated CD59. Thus, measuring the levelof glycated CD59 may be carried out by measuring the relative amount ofnonglycated CD59. Similarly, when determining the level of glycated CD59in a sample using the mass spectrometric or other peptide identificationmethod of the invention, the difference between the total amount of CD59and the amount of non-glycated CD59 after digestion of the spiked sampleis the amount of glycated CD59 in the sample.

It will also be understood that the controls according to the inventionmay be, in addition to predetermined values, samples of materials testedin parallel with the experimental materials. Examples include samplesfrom control populations or control samples generated throughmanufacture to be tested in parallel with the experimental samples.

In some embodiments of the invention, methods provided are used todetermine the level of glycated CD59 is a subject at risk of having adiabetic disorder, or blood sugar regulation disorder. As used herein, asubject “at risk” is a subject who is considered more likely to developa disease state or a physiological state than a subject who is not atrisk. A subject “at risk” may or may not have detectable symptomsindicative of the disease or physiological condition, and may or may nothave displayed detectable disease prior to the treatment methods (e.g.,therapeutic intervention) described herein. “At risk” denotes that asubject has one or more so-called risk factors. A subject having one ormore of these risk factors has a higher probability of developing one ormore disease(s) or physiological condition(s) than a subject withoutthese risk factor(s). These risk factors can include, but are notlimited to, history of family members developing one or more diseases(e.g. diabetes), related conditions (e.g. pregnancy), or pathologies,history of previous disease, age, sex, race, diet, presence of precursordisease, genetic (i.e., hereditary) considerations, and environmentalexposure. The level of risk can be assessed using standard methods knownto those in the art. For example, based on factors such as medicalhistory, family medical history, and current medical condition, a healthcare professional may assess a percentage chance that a subject willhave or will develop a diabetic disorder or blood sugar regulationdisorder. For example, a health care professional may determine that asubject who had gestational diabetes or borderline gestational diabetesmay have a 20%, 30%, 40%, 50%, 60%, 70% or more chance of developinggestational diabetes in a subsequent pregnancy. Those of skill in theart will recognize that a subject's level of risk for other diabeticdisorders or blood sugar regulation disorders can also be evaluatedusing standard methods.

As mentioned above, it is also possible to characterize blood sugarlevels by monitoring changes in the absolute or relative amounts ofglycated CD59 over time. For example, it is expected that an increase inglycated CD59 correlates with increasing dysregulation of glycemiclevels. Accordingly one can monitor glycated CD59 levels over time todetermine if glycemic levels of a subject are changing. Changes inrelative or absolute glycated CD59 of greater than 0.1% may indicate anabnormality. Preferably, the change in glycated CD59 levels, whichindicates an abnormality, is greater than 0.2%, greater than 0.5%,greater than 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%,30%, 40%, 50%, or more. Reductions in amounts of glycated CD59 over timemay indicate improved glycemic control. For example, reductions in theamount of glycated CD59 of greater than 0.2%, greater than 0.5%, greaterthan 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%, 30%, 40%,50%, or more indicate increased glycemic control in a subject.

The invention in another aspect provides a diagnostic method todetermine the effectiveness of treatments for abnormal glycemic levels.The “evaluation of treatment” as used herein, means the comparison of asubject's levels of glycated CD59 measured in samples collected from thesubject at different sample times, preferably at least one day apart.The preferred time to obtain the second sample from the subject is atleast one day after obtaining the first sample, which means the secondsample is obtained at any time following the day of the first samplecollection, preferably at least 12, 18, 24, 36, 48 or more hours or daysafter the time of first sample collection. In some embodiments the timebetween sample collections is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ormore hours.

The comparison of levels of glycated CD59 in two or more samples, takenat different times, or on different days, is a measure of level of thesubject's glycemic control and allows evaluation of the treatment toregulate blood sugar levels. The comparison of a subject's levels ofglycated CD59 measured in samples obtained at different times or ondifferent days provides a measure of glycemic control to determine theeffectiveness of any treatment to regulate blood sugar levels.

As will be appreciated by those of ordinary skill in the art, theevaluation of the treatment also may be based upon an evaluation of thesymptoms or clinical end points of the associated disease, such as thevascular complications of diabetes. Thus, the methods of the inventionalso provide for determining the onset, progression, and/or regressionof a condition which is characterized by abnormal levels of glycatedprotein, including those characterized by abnormal levels of glycatedCD59. In some instances, the subjects to which the methods of theinvention are applied are already diagnosed as having a particularcondition or disease. In other instances, the measurement will representthe diagnosis of the condition or disease. In some instances, thesubjects will already be undergoing drug therapy for regulating bloodsugar levels, while in other instances the subjects will be withoutpresent drug therapy for regulating blood sugar levels.

According to still another aspect of the invention, a method is providedfor treating a subject to reduce the risk of a disorder associated withabnormally high levels of glycated CD59. The method involves selectingand administering to a subject who is known to have an abnormally highlevel of glycated CD59, an agent for treating the disorder. Preferably,the agent is an agent for reducing glycated CD59 levels and isadministered in an amount effective to reduce glycated CD59 levels.

In this aspect of the invention, the treatments are based upon selecting(e.g. identifying) subjects who have unwanted, elevated levels ofglycated CD59. Such subjects may already be receiving a drug forregulating blood sugar levels, but using the methods of the invention,are now identified as candidates for an elevated level of the drug basedupon the presence of the elevated levels of glycated CD59. It may beappropriate according to the invention to alter a therapeutic regimenfor a subject, based upon the measurement of the level of glycated CD59.This can be understood in connection with treatment of diabetics.

Diabetics are treated in at least three different ways. Some diabeticsare treated only with non-drug therapy, such as exercise and diet. Otherdiabetics are treated with oral drug therapy, but not with insulin thatis injected. Finally, some diabetics are treated with insulin or analogsof insulin by injection. According to the present invention, as a resultof determining an elevated level of glycated CD59, an individualundergoing only non-drug therapy may be a candidate for drug therapy asa result of the glycated CD59 test. Likewise, a subject receiving onlyoral drug therapy may be a candidate for an insulin-based injectabletherapy, due to the glycated CD59 test. Finally, a subject may be freeof any present treatment but may be a candidate for blood sugar levelregulating treatment as a result of the glycated CD59 test. Thus,subjects may be selected (e.g. identified) and treated with elevatedlevels of the same drugs or with different therapies as a result of theassays of the invention.

According to the present invention, some of the subjects are free ofsymptoms otherwise calling for treatment with a particular therapy. Thismeans that absent the glycated CD59 test, the subject would notaccording to convention as of the date of the filing of the presentapplication have symptoms calling for treatment with a particulartherapy. It is only as a result of the measuring the level of glycatedCD59 that the subject becomes a candidate for treatment with thetherapy.

Drug therapies for regulating blood sugar levels include oral therapieswith hypoglycemic agents and/or oral anti-diabetic agents, injectabletherapies, and the like. Non-drug therapies for regulating blood sugarlevel include, but are not limited to, diatetic and/or exercise controlmeasures.

Diet and exercise alterations include, but are not limited to, reducingcaloric intake, and/or increasing fiber intake, and/or decreasing fatintake, and/or increasing exercise level.

Oral drug therapies for regulating blood sugar levels includehypoglycemic agents that may include, but are not limited to:

Acarbose; Acetohexamide; Chlorpropamide; Darglitazone Sodium:Glimepiride; Glipizide; Glyburide, Repaglinide; Troglitazone;Tolazamide; Tolbutamide.

Oral drug therapies for regulating blood sugar levels includeantidiabetic agents that may include but are not limited to: Acarbose,Acetohexamide; Buformin; Butoxamine Hydrochloride; Camiglibose;Chlorpropamide; Ciglitazone; Englitazone Sodium; EtoforminHydrochloride; Gliamilide; Glibornuride; Glicetanile, Gliclazide Sodium;Gliflumide; Glipizide; Glucagon; Glyburide; Glyhexamide; GlymidineSodium; Glyoctamide; Glyparamide; Insulin; Insulin, Dalanated; InsulinHuman; Insulin Human, Isophane; Insulin Human Zinc; Insulin Human Zinc,Extended; Insulin, Isophane; Insulin Lispro; Insulin, Neutral; InsulinZinc; Insulin Zinc, Extended; Insulin Zinc, Prompt; Linogliride;Linogliride Fumarate; Metformin; Methyl Palmoxirate; Palmoxirate Sodium;Pioglitazone Hydrochloride; Pirogliride Tartrate; Proinsulin Human;Repaglinide; Seglitide Acetate; Tolazamide; Tolbutamide; Tolpyrramide;Troglitazone; Zopolrestat.

Injectable therapies for regulating blood sugar levels include, but arenot limited to: Fast-Acting Insulin:

Insulin Injection: regular insulin; Prompt Insulin Zinc Suspension;Semilente® insulin. These categories include preparations such as:Humalog® Injection; Humulin® R; Iletin II; Novolin R, Purified PorkRegular Insulin; Velosulin BR Human Insulin Intermediate-acting Insulin:

Isophane Insulin Suspension: NPH insulin, isophane insulin; Insulin ZincSuspension Lente® Insulin. These categories include preparations suchas: Humulin® L; Humulin® R; Humulin® N NPH; Iletin® II, Lente®; Iletin®II, NPH; Novolin® L, Novolin® N, Purified Pork Lente® insulin, PurifiedPork NPH isophane insulin.

Intermediate and Rapid-Acting Insulin Combinations:

Human Insulin Isophane Suspension/Human Insulin Injection. This categoryincludes preparations such as: Humulin® 50/50; Humulin®70/30;Novolin®70/30 Long-acting Insulin: Protamine Zinc Insulin Suspension;Extended Insulin Zinc Suspension. These categories include preparationssuch as: Ultralente® Insulin, Humulin® U.

Reducing the risk of a disorder associated with abnormally high levelsof glycated CD59 means using treatments and/or medications to reduceglycated CD59 levels, therein reducing, for example, the subject's riskof vascular complications including but not limited to: diabeticnephropathy, diabetic retinopathy, macro-vascular disease,micro-vascular disease, and diabetic neuropathy.

In a subject determined to have an abnormally high level of glycatedCD59, an effective amount is that amount effective to reduce glycatedCD59 levels in the subject. A response can, for example, also bemeasured by determining the physiological effects of the hypoglycemic,antidiabetic, or insulin composition, such as the decrease of diseasesymptoms following administration of the hypoglycemic, antidiabetic, orinsulin. Other assays will be known to one of ordinary skill in the artand can be employed for measuring the level of the response. The amountof a treatment may be varied for example by increasing or decreasing theamount of a therapeutic composition, by changing the therapeuticcomposition administered, by changing the route of administration, bychanging the dosage timing and so on. The effective amount will varywith the particular condition being treated, the age and physicalcondition of the subject being treated, the severity of the condition,the duration of the treatment, the nature of the concurrent therapy (ifany), the specific route of administration, and the like factors withinthe knowledge and expertise of the health practitioner. For example, aneffective amount can depend upon the degree to which an individual hasabnormally elevated levels of glycated CD59.

An “effective amount” of a drug therapy is that amount of ahypoglycemic, antidiabetic, or insulin or insulin analog that alone, ortogether with further doses, produces the desired response, e.g.reduction of glycemic level or glycated CD59 levels.

In the case of treating a particular disease or condition the desiredresponse is inhibiting the progression of the disease or condition. Thismay involve only slowing the progression of the disease temporarily,although more preferably, it involves halting the progression of thedisease permanently. This can be monitored by routine diagnostic methodsknown to one of ordinary skill in the art for any particular disease.The desired response to treatment of the disease or condition also canbe delaying the onset or even preventing the onset of the disease orcondition.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of the hypoglycemic, antidiabetic, or insulincomposition (alone or in combination with other therapeutic agents) beused, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of hypoglycemic,antidiabetic, or insulin for producing the desired response in a unit ofweight or volume suitable for administration to a patient.

The doses of hypoglycemic, antidiabetic, or insulin administered to asubject can be chosen in accordance with different parameters, inparticular in accordance with the mode of administration used and thestate of the subject. Other factors include the desired period oftreatment. In the event that a response in a subject is insufficient atthe initial doses applied, higher doses (or effectively higher doses bya different, more localized delivery route) may be employed to theextent that patient tolerance permits.

Various modes of administration will be known to one of ordinary skillin the art which effectively deliver the hypoglycemic, antidiabetic, orinsulin to a desired tissue, cell or bodily fluid. Preferred methods foradministering the hypoglycemic and antidiabetic are oral. The preferredmethod of administering insulin is by injection. Administrationincludes: topical, intravenous, oral, intracavity, intrathecal,intrasynovial, buccal, sublingual, intranasal, transdermal,intravitreal, subcutaneous, intramuscular and intradermaladministration. The invention is not limited by the particular modes ofadministration disclosed herein. Standard references in the art (e.g.,Remington's Pharmaceutical Sciences, 18th edition, 1990) provide modesof administration and formulations for delivery of variouspharmaceutical preparations and formulations in pharmaceutical carriers.Other protocols which are useful for the administration of hypoglycemic,antidiabetic, or insulin will be known to one of ordinary skill in theart, in which the dose amount, schedule of administration, sites ofadministration, mode of administration (e.g., intra-organ) and the likevary from those presented herein.

Administration of hypoglycemic, antidiabetic, or insulin to mammalsother than humans, e.g. for testing purposes or veterinary therapeuticpurposes, is carried out under substantially the same conditions asdescribed above. It will be understood by one of ordinary skill in theart that this invention is applicable to both human and animal diseaseswhich can be treated by hypoglycemic, antidiabetic or insulin. Thus,this invention is intended to be used for diagnostics and therapeuticsin husbandry and veterinary medicine as well as for human diagnosticsand therapeutics.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts. Preferred components of the composition aredescribed above in conjunction with the description of the hypoglycemic,antidiabetic, or insulin compositions of the invention.

A hypoglycemic, antidiabetic, or insulin composition may be combined, ifdesired, with a pharmaceutically-acceptable carrier. The term“pharmaceutically acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the hypoglycemic, antidiabetic, or insulin, andwith each other, in a manner such that there is no interaction whichwould substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,as described above, including: acetate, phosphate, citrate, glycine,borate, carbonate, bicarbonate, hydroxide (and other bases) andpharmaceutically acceptable salts of the foregoing compounds.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise hypoglycemid, antidiabetic, or insulin. This preparation may beformulated according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationalso may be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

The application of the invention to a diabetic subject under treatmentwith an oral blood sugar regulating agent and otherwise free of symptomscalling for any oral blood sugar regulating agent, as used herein meansa subject treated with oral blood sugar regulators whoseglycemic-control levels appear normal based on standard diagnosticcriteria, including but not limited to measurement of glycatedhemoglobin levels.

The application of the invention to a diabetic subject under treatmentwith insulin (including analogs thereof) and otherwise free of symptomscalling for any insulin, as used herein means a subject treated withinsulin whose glycemic-control levels appear to be normal based onstandard diagnostic criteria, including but not limited to measurementof glycated hemoglobin levels.

Dosages of blood sugar regulating agents are well-known to those ofordinary skill in the art and documented in the literature.

Also within the scope of the invention are kits comprising thecompositions of the invention and instructions for use. The kits canfurther contain at least one additional reagent, such as a controlsample. Kits of the invention can be prepared for in vitro diagnosis,prognosis and/or monitoring a diabetic condition, blood sugar regulatingdisorder, or complication by the mass spectrometry and other detectionmethods described above. Kits of the invention may include a CD59peptide and the CD59 peptide may be a native or labeled CD59 peptide.

For example, kits containing a CD59₍₂₉₋₅₄₎ peptideTKAGLQVYNKCWKFEHCNFNDVTTRL; (SEQ ID NO:5) that includesdeuterated-(d₈)-V₍₃₅₎ and d₈-F₍₄₇₎ can be prepared. The components ofthe kits can be packaged either in aqueous medium or in lyophilizedform. A kit of the invention, in some embodiments, may further comprisea container containing glycated CD59 molecule, which may be K41 glycatedCD59 and/or a container containing non-glycated CD59 or non-K41 glycatedCD59. Some or all of the kit components may be frozen.

A kit of the invention may also include a container containing anenzyme, for example, trypsin. The enzyme may be in lyophilized form ormay be in aqueous medium. The kit may also include the solution forreconstitution of the enzyme. A kit of the invention may also includecontrol compounds and solutions for testing the activity of the enzyme,for example materials to perform an enzymatic assay of trypsin. Suchmaterials may include, buffer, a non-limiting example of which is sodiumphosphate buffer, Nα-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE), HClor other acid and trypsin. A kit may also include instructions fordetermining the activity of the enzyme per unit of the enzyme.

A kit of the invention may comprise a carrier being compartmentalized toreceive in close confinement therein one or more container means orseries of container means such as test tubes, vials, flasks, bottles,syringes, or the like. A first of said container means or series ofcontainer means may contain a CD59 peptide, such as, but not limited, toCD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO:5) thatincludes deuterated-(d₈)-V₍₃₅₎ and d₈-F₍₄₇₎. A second container means orseries of container means may contain glycated CD59 and/or non-glycatedCD59. A third container may include the enzyme, e.g. trypsin. In someembodiments, the kit may include one or more solutions or molecules thatcan be used to make a labeled CD59 peptide. An example of a labeled CD59peptide, though not intended to be limiting, is CD59 peptide that isdeuterated-(d₈) at V₍₃₅₎ and F₍₄₇₎ (corresponding to the residues ofmature CD59). A kit of the invention may also include a native(unlabeled) CD59 peptide. For example, a kit of the invention mayinclude native CD59₍₂₉₋₅₄₎ peptide TKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ IDNO:11), or other native CD59 peptide. One of ordinary skill in the artwill recognize that in addition to deuterated peptides, peptides of thekits may be labeled with other types of labels and the kits may includeadditional types of labeling compounds.

A kit of the invention may also include an antibody that specificallybinds to a CD59 peptide. For example, a kit may include an antibody thatspecifically recognizes a trypsin digest fragment of CD59. Such anantibody can be used to detect the presence of digest fragments usingthe methods provided herein, thus allowing the determination of thepresence and/or level of glycated CD59 in a sample.

A kit of the invention may also include vials, cuvettes, pipet tips,transfer pipets, solutes, sterile and/or distilled water, one or morecontrol samples, (e.g. blank control, test control), printed graphs,tables, figures, or diagrams, which may be used for interpretationand/or analysis of results or for instructional purposes.

A kit of the invention may also include equipment and/or supplies formass spectrometric or other methods of determining the level of glycatedCD59. For example, a kit may include ELISA assay materials, gelpreparation materials (e.g. solutions, agarose, to acrylamide, controlmarkers, dyes and/or labels, etc,). A kit may also include materials forchromatographic analysis, e.g. beads, solvents, solutes, control samplesetc, columns, etc.

In some embodiments, materials for analysis of the level of glycatedCD59 are provided in a ready-to-use format. In other embodiments, thekits provide materials that can be utilized for determining the level ofglycated CD59 in a sample and will be assembled for use by the operator.Some kits of the invention will include all materials necessary fordetermining the level of glycated CD59 in a sample, and other kits ofthe invention will include some, but not all of the materials for thedetermination of the level of CD59 in a sample. In the latter case,additional materials will be provided by the operator and may include:gas chromatographs, mass spectrophotometers, pipets, tubes, gelapparatus, flasks, solutions, enzymes, CD59, etc.

Referring to FIG. 1, a kit according to the invention is shown. The kit11 includes a package 15 housing a container 17 which contains an agentfor determining the level of glycated CD59 in a sample. The kit alsoincludes a control 19. The kit also may further comprise instructions21, as described above. The instructions typically will be in writtenform and will provide guidance for carrying-out the assay embodied bythe kit and for making a determination based upon that assay.

Examples Introduction

Mass spectrometry is a highly accurate and reproducible method that iscurrently being used to quantitate small molecules in plasma and urinesamples. This methodology allows the analysis of a large number ofsamples in high throughput mode. The relative high levels of solubleCD59 in urine (5 μg/ml) and plasma (15-20 ng/ml) favor the feasibilityof this approach. Trypsin hydrolyzes peptide bonds on the N-terminus ofpositively charged amino acids such as K and R. Our studies confirmedthat modifications such as glycation of the amino acid side chainchanged the trypsin peptide map of proteins, hence, glycated K41 on CD59is no longer recognized as a trypsin cut site.

Methods

For mass spectrometry analysis, we have synthesized the CD59₍₂₉₋₅₄₎peptide TKAGLQVYNKCWK*FEHCNFNDVTTRL (SEQ ID NO:5) withdeuterated-(d₈)-V₍₃₅₎ and d₈-F₍₄₇₎. A known amount of this peptide wasadded as an internal standard to each sample prior to trypsinization.The spiked samples were then trypsinized for 30 minutes at 37° C.Trypsinization of the urine or plasma sample with the added internalstandard d₈-CD59₍₂₉₋₅₄₎ peptide yielded the following peptides:

TABLE 1 Trypsin products of non-glycated CD59. Trypsin products ofTrypsin products of  non-glycated CD59  CD59₍₂₉₋₅₄₎ peptideAGLQVYNK (SEQ ID NO: 8) AGLQV_(d8)YNK (SEQ ID NO: 6) CWK CWKFEHCNFNDVTTR  FEHCNF_(d8)NDVTTR  (SEQ ID NO: 9) (SEQ ID NO: 7)

In contrast: trypsin digestion of glycated CD59 yielded only twopeptides because glycated CD59 is not cleaved at the glycated K41 site(*=K41 site).

TABLE 2 Trypsin products of glycated CD59Trypsin products of glycated CD59 AGLQVYNK (SEQ ID NO: 8)CWKFEHCNFNDVTTR (SEQ ID NO: 10)

Because isotopically labeled and natural peptides ionize in an identicalmanner, the ionization of the internal standard peptides [AGLQV_(d8)YNK(SEQ ID NO:6) and FEHCNF_(d8)NDVTTR (SEQ ID NO:7)] was identical to theionization of the peptides derived from the subject's CD59 [AGLQVYNK(SEQ ID NO:8) and FEHCNFNDVTTR (SEQ ID NO:9)]. Thus, peptides derivedfrom either the internal standard or the subject's CD59 have identicalcharge but a different mass of 8 units that allows their unambiguousidentification and quantitation provided that the amount of the labeledCD59₍₂₉₋₅₄₎ peptide added to the sample was known. The mass differenceof 8 is the result of the presence of V_(d8) in AGLQV_(d8)YNK (SEQ IDNO:6) and of F_(d8) in FEHCNF_(d8)NDVTTR (SEQ ID NO:7).

The AGLQVYNK (SEQ ID NO:8) peptide was generated by the trypsindigestion of both glycated and non-glycated CD59. Thus, the quantitativeestimate of AGLQVYNK (SEQ ID NO:8) represented total CD59 in the sample.In contrast, the FEHCNFNDVTTR (SEQ ID NO:9) peptide was only generatedby the trypsin digestion of non-glycated CD59. Thus, the quantitativeestimate of FEHCNFNDVTTR (SEQ ID NO:9) represented non-glycated CD59 inthe sample. The difference between total and non-glycated CD59represented the amount of glycated CD59.

This method was less cumbersome and more accurate than the standardELISA and thus allows processing of hundreds of samples per day.

Results

Progress on the use of isotope ration mass spectrometry for thequantitation of CD59 and glycated CD59.

1) The LCQ Advantage with an APCI probe was switched over to the ESIprobe and tuned on MRFA.2) The instrument was tuned and a tune file was created using thetryptic digest of the isotopically labeled peptideTKAGLQVYNKCWKFEHCNFNDVTTRL (SEQ ID NO: 5).3) a test run was performed on the LCMS on the tryptic digested peptideusing the Biobasic 100 column for the LC.FIG. 2 is a graph of the total ion current of the peptide.FIG. 3 is the mass spectrometry profile showing the peptides ofinterest. (AGLQV is SEQ ID NO:12; QVYNK is SEQ ID NO:13; andFEHCNFNDVTTR is SEQ ID NO:9).

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose and variations can be made by those skilled in the art withoutdeparting from the spirit and scope of the invention which is defined bythe following claims.

The contents of all references, patents, patent documents, and publishedpatent applications cited throughout this application are incorporatedherein by reference in their entirety.

1. A method for assessing the onset, progression, or regression of acondition characterized by abnormal levels of glycated proteincomprising: obtaining a level of glycated CD59 from a sample obtainedfrom a subject; and comparing the level to a control as an assessment ofthe onset, progression, or regression of the condition.
 2. The method ofclaim 1, wherein the step of obtaining a level of glycated CD59comprises: digesting CD59 in the sample; identifying a peptide yieldedby the digestion of the CD59 in the sample; and quantitating theidentified peptide as a determination of the level of glycated CD59 inthe sample.
 3. The method of claim 2, wherein the digestion is anenzymatic digestion.
 4. The method of claim 2, wherein the enzyme istrypsin.
 5. The method of claim 2, wherein the peptide is identifiedusing immunoassay, gel electrophoresis, NMR, Western blotting,chromatography, or mass spectrometry.
 6. The method of claim 2, whereinthe peptide is identified using mass spectrometry.
 7. The method ofclaim 2, wherein the peptide is quantitated by comparing the level ofthe peptide to the level of one or more additional peptides yielded bythe digestion.
 8. The method of claim 2, wherein the peptide isquantitated by comparing the level of the peptide to a control level. 9.The method of claim 2, wherein the peptide identified is a peptide withone or more labels.
 10. The method of claim 2, wherein the peptideidentified is a peptide having an amino acid sequence set forth asAGLQVYNK (SEQ ID NO:8), CWK, FEHCNFNDVTTR (SEQ ID NO:9), orCWKFEHCNFNDVTTR (SEQ ID NO:10).
 11. The method of claim 2, furthercomprising spiking the sample with an internal standard beforedigestion.
 12. The method of claim 11, wherein the internal standard hasone or more labels.
 13. The method of claim 11, wherein the internalstandard is a CD59 peptide.
 14. The method of claim 13, wherein theinternal standard is a peptide comprising the amino acid sequence setforth as AGLQVYNKCWKFEHCNFNDVTTR (SEQ ID NO:15).
 15. The method of claim14, wherein the one or more labels are on an amino acid of the portionof the sequence set forth as SEQ ID NO:15 that is AGLQVYNK (SEQ IDNO:8).
 16. The method of claim 14, wherein the one or more labels are onan amino acid of the portion of the sequence set forth as SEQ ID NO:15that is CWKFEHCNFNDVTTR (SEQ ID NO:10).
 17. The method of claim 14,wherein the one or more labels are on an amino acid of the portion ofthe sequence set forth as SEQ ID NO:15 that is CWK.
 18. The method ofclaim 14, wherein the one or more labels are on an amino acid of theportion of the sequence set forth as SEQ ID NO:15 that is FEHCNFNDVTTR(SEQ ID NO:9).
 19. The method of claim 14, wherein one label is on anamino acid of the portion of SEQ ID NO:15 that is AGLQVYNK (SEQ IDNO:8), and one label is on an amino acid of the portion of SEQ ID NO:15that is FEHCNFNDVTTR (SEQ ID NO:9).
 20. The method of claim 11, whereinthe peptide is quantitated by comparing the level of the peptidedetermined in the spiked sample, the level of the internal standardadded to the sample, and the level of one or more additional peptidesyielded by the digestion.
 21. The method of claim 11, wherein thepeptide is quantitated by comparing the level of the peptide to acontrol level.
 22. The method of claim 20, wherein the peptidequantitated is a peptide having the amino acid sequence set forth asAGLQVYNK (SEQ ID NO:8), CWK, FEHCNFNDVTTR (SEQ ID NO:9), orCWKFEHCNFNDVTTR (SEQ ID NO:10).
 23. The method of claim 20, wherein thepeptide quantitated is labeled with one or more labels.
 24. The methodof claim 20, wherein the labeled peptide is AGLQVYNK (SEQ ID NO:8) orFEHCNFNDVTTR (SEQ ID NO:9).
 25. The method of claim 24, wherein the oneor more labels are on one or more of the amino acids of the sequence setforth as AGLQVYNK (SEQ ID NO:8).
 26. The method of claim 24, wherein theone or more labels are on one or more of the 48 acids of the sequenceset forth as FEHCNFNDVTTR (SEQ ID NO:9).
 27. The method of claim 2,wherein the sample is a fluid sample.
 28. The method of claim 27,wherein the fluid sample is blood, urine, or saliva.
 29. The method ofclaim 2, wherein the sample is a tissue sample.
 30. The method of claim1, wherein the subject is diabetic.
 31. The method of claim 1, whereinthe subject is at increased risk of becoming diabetic.
 32. The method ofclaim 1, wherein the subject has received treatment for regulating bloodsugar levels.
 33. The method of claim 1, wherein the subject has notreceived treatment for regulating blood sugar levels.